朱砂根和树豆叶的化学成分及抗肿瘤作用研究
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摘要
恶性肿瘤是当前危害人类健康的重大疾病之一。其死亡率之高,已跃居所有死亡病因的第二位。肿瘤治疗的主要手段是化学药物的治疗,简称化疗。肿瘤化疗药物主要有合成药物和天然药物。合成的抗肿瘤药物中,许多都是以天然抗肿瘤活性成分为先导化合物发展起来的,我国的药用植物资源丰富,从中草药中寻找有效的天然抗肿瘤活性成分作为肿瘤化疗药物先导化合物,具有明显的优势。
本学位论文旨在研究发现天然抗肿瘤药物活性先导化合物。由两部分组成。
第一部分朱砂根中百两金皂苷A的分离鉴定、批量制备及抗肿瘤作用研究
研究背景和目的
作者通过前期的试验和文献调研,认为中药朱砂根(Ardisia crenata Sims.)中的三萜皂苷类化合物百两金皂苷A、B (ardisiacrispin A, B)及其结构类似物具有明显的抗肿瘤药效；鉴于文献主要集中在朱砂根皂苷的体外抗肿瘤药理研究方面,本课题对朱砂根三萜皂苷类化合物进行分离鉴定,开展毒理学和体外体内抗肿瘤活性研究,为考察百两金皂苷A类似物作为抗肿瘤药物先导化合物的可行性提供实验依据。
研究方法
(1)利用正相硅胶、MCI、反相ODS等柱层析手段,从朱砂根的正丁醇部位中分离皂苷类化合物；采用MS、1H-NMR、13C-NMR等波谱学方法鉴定化学结构。
(2)采用MTT法检测百两金皂苷A对多种人肿瘤细胞株和人非肿瘤细胞株的细胞毒性作用,利用瑞氏-吉姆萨染色法和流式细胞仪检测了百两金皂苷A对HCT-15细胞凋亡和细胞周期的影响。
(3)首次完成了化合物百两金皂苷A纯品的急性毒性和亚急性毒性研究。
(4)采用人HCT-15肿瘤裸鼠皮下移植模型,对化合物百两金皂苷A开展了体内抗肿瘤作用试验。
研究结果
(1)从朱砂根中分离得到3个化合物,其化学结构鉴定为：百两金皂苷A(ardisiacrispin A)、朱砂根皂苷B(ardisicrenoiside B)和岩白菜素(Bergenin)。分离制备了多达45g的百两金皂苷A纯品作为体内实验研究用药。
(2)体外抗肿瘤试验结果表明,百两金皂苷A对人非小细胞肺癌细胞株NCI-H460、人前列腺癌细胞株PC-3、人乳腺癌细胞株MCF-7、人宫颈癌细胞株Hela.人结直肠腺癌细胞株HCT-15、人口腔上皮癌耐药细胞株KB-V1等肿瘤细胞均具有明显的增殖抑制作用,其半数抑制浓度(IC5o)分别为7.61μM、8.29μM、8.64μM、5.30μM和4.05μM；对人肝静脉内皮细胞株ED-25的细胞毒性(IC50为8.11μM),与对上述肿瘤细胞的细胞毒性无显著差异(P>0.05),提示百两金皂苷A的细胞毒作用可能无选择性。其中,百两金皂苷A对HCT-15细胞的体外研究结果系首次发表。经细胞形态学观察发现,8μM的百两金皂苷A处理后,HCT-15细胞出现早期凋亡样形态学改变：细胞变圆,核染色质浓集,固缩,碎裂。推测其细胞毒性可能与诱导凋亡有关。Annexin V/PI双染法检测试验结果表明,随着百两金皂苷A药物浓度的增加,HCT-15细胞平均早期凋亡率逐渐上升,并呈一定的浓度依赖性。结合形态学观察结果,表明百两金皂苷A对HCT-15的细胞毒作用是通过诱导凋亡产生的。流式细胞仪检测发现,经百两金皂苷A处理可以引起HCT-15细胞发生S期阻滞。因此,百两金皂苷A可能是藉此诱导HCT-15细胞凋亡的。
(5)急性毒性试验结果显示,百两金皂苷A对昆明种小鼠的半数致死量LD50为1.44g/kg,属于低毒化合物。亚急性毒性试验中,连续灌胃4周造成了大鼠轻微肝损伤,肝细胞轻度嗜酸性变,细胞固缩,连接松散。预示着百两金皂苷A对肝脏可能有一定影响。
(6)在人HCT-15肿瘤裸鼠移植瘤模型实验中,以60mg/kg、50mg/kg、40mg/kg剂量灌胃给药百两金皂苷A,与阳性药多西他赛组相比,相对肿瘤增殖率AT/AC大于42%,根据NCI评判标准,认为该途径下百两金皂苷A对HCT-15人癌裸鼠模型的抗肿瘤药效不明显。
结论与创新点
(1)从朱砂根中分离得到3个化合物,经波谱学技术鉴定为百两金皂苷A、B,以及岩白菜素；制备了多达45g的百两金皂苷A纯品,作为体内实验研究用药。
(2)首次研究报告了百两金皂苷A对HCT-15细胞的细胞增殖抑制作用；该作用的机理与诱导肿瘤细胞凋亡和阻滞细胞增殖于S期有关。
(3)首次研究评价了百两金皂苷A的急性、亚急性毒性。结果表明,该化合物口服低毒。
(3)在人HCT-15肿瘤细胞裸鼠皮下移植模型上,测试了灌胃给药条件下百两金皂苷A的体内抗肿瘤作用,结果表明作用不明显。
第二部分树豆叶的化学成分及抗肿瘤作用研究
研究背景、目的
作者通过预实验和文献调研,认为树豆(Cajanus cajan (L.) Millsp.)叶中木豆素A、C (longistylin A, C)的二苯乙烯(菧)结构类似物有明显的抗肿瘤作用,而且目前对树豆蔗类成分的研究尚不够系统和深入。因此,课题组对树豆叶的菧类化合物进行了较为系统的分离和结构鉴定研究、体外抗肿瘤活性与机理研究,以期获得新菧类抗癌先导化合物。
研究方法
(1)利用『正相硅胶、Sephadex LH-20凝胶等柱层析手段,从树豆的三氯甲烷部位中分离蔗类化合物,并采用IR、1H-NMR、13C-NMR、2D-NMR等波谱学技术,结合EI-MS、ESI-MS和HRESI-MS等质谱和元素分析进行综合分析,鉴定化学结构。
(2)采用MTT法检测分离得到的菧类化合物对多种人肿瘤细胞株和人非肿瘤细胞株的细胞毒性作用,利用瑞氏-吉姆萨染色法和流式细胞仪检测菧类化合物对肿瘤细胞的凋亡和细胞周期的影响。
研究结果
(1)从树豆中分离得到7个化合物(1-7)。它们的化学结构经波谱学分析鉴定为：木豆素A(longistylin A)(1)、木豆素C(longistylin C)(2)、树豆菧H (cajanstilbene H)(3)、树豆内酯A (cajanolactone A)(4)等4个蔗类化合物；异美五针松双氢黄酮(pinostrobin)(5)、柚皮素4’,7-二甲醚(naringenin-4',7-dimethyl ether)(6)等2个黄酮；以及1个甾类化合物β-谷甾醇(β-sitosterol)(7)。其中3和4为新化合物,分别命名为树豆菧H (cajanstilbene H)和树豆内酯A (cajanolactone A),前者是从该属植物中首次发现的含卤素的羟基异戊烯取代二苯乙烯衍生物,后者为该属植物中首次发现的内酯化的羟基异戊烯取代二苯乙烯羧酸衍生物。
(2)四种菧类化合物树豆菧H (cajanstilbene H)、树豆内酯A (cajanolactone A)、 longistylin A、longistylin C对NCI-H460、PC-3、MCF-7、Hela、HCT-15、KB-V1等人源肿瘤细胞株的增殖表现出明显抑制作用。
其中新化合物cajanstilbene H对上述肿瘤细胞的半数抑制浓度IC5o分别为21.47μM、25.83μM、21.42μM、25.85μM、24.81μM、22.29μM;对人肝静脉内皮细胞株ED-25的IC5o为38.51μM,小于对肿瘤细胞的细胞毒性,差异有显著性(P<0.05)。
细胞形态学观察发现,经42μM的cajanstilbene H处理后,NCI-H460细胞出现早期凋亡样形态学改变：细胞变圆,核染色质浓集,核固缩,碎裂。推测其细胞毒性也可能与诱导凋亡有关。Annexin V/PI双染法检测结果表明,随着cajanstilbene H药物浓度的增加,NCI-H460细胞平均早期凋亡率逐渐上升,并呈一定的浓度依赖性。说明cajanstilbene H对NCI-H460细胞的细胞毒作用是通过诱导凋亡产生的。
经流式细胞仪检测,cajanstilbene H将NCI-H460细胞能阻滞于G0/G1期而抑制增殖。
结论与创新点
(1)从树豆叶中发现了2个新的菧类化合物,其结构系通过详细的波谱学综合分析所确定,分别命名为树豆菧H (cajanstilbene H)和树豆内酯A (cajanolactone A),前者是从该属植物中首次发现的含卤素的羟基异戊烯取代二苯乙烯衍生物,后者为该属植物中首次发现的内酯化的羟基异戊烯取代二苯乙烯羧酸衍生物。
(2)研究了新化合物树豆菧H (cajanstilbene H)、树豆内酯A (cajanolactone A)、和结构类似物longistylin A、longistylin C对NCI-H460、PC-3、MCF-7、Hela、HCT-15、 KB-V1等肿瘤细胞株的体外作用,发现新化合物具有明显的体外抗肿瘤作用。初步揭示了新化合物树豆菧H的体外抗肿瘤作用机理,发现树豆菧H可诱导NCI-H460细胞的凋亡,并使细胞周期阻滞于G0/G1期。
Malignant tumor or cancer is one of the major diseases that damage severely human health, and is the second leading cause of death in the world. Chemical medication is the major treatment method for cancer at present. Chemotherapeutic agents mainly contain synthetic and natural medicines. Many of synthetic drugs are of natural origin or derived from the structures of natural products. China is rich in medicinal plant resources, so it is a decided advantage on development of safe and effective anticancer drugs or lead compounds from medicinal herbs. The aim of this dissertation is to search natural anticancer leading compounds. It consists of two parts.
Part One Ardisiacrispin A from Ardisia crenata:Isolation, Identification, Lot Manufacture, and Anticancer Effect
Background and Objective
According to our previous experiments and literatures, ardisiacrispin A, B from Ardisia crenata Sims., as well as their derivatives, exhibited significant anticancer activity They are worthy of our further and systemaic researches, including phytochemical study, in vitro and in vivo pharmacological studies for searching of anticancer leading compounds
Method
(1) Ardisiacrispin A and relavent constituents were isolated from the n-BuOH extract of Ardisia crenata by silica gel, MCI and ODS column chromatographies. The structures were elucidated on the basis of spectroscopic analysis, mainly on MS and NMR.
(2) Effects of ardisiacrispin A on cancer cell lines were examined by MTT assay. The apoptosis was observed by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.
(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests.
(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was also carried out.
Result
(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.
(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines and cell line ED-25, human liver vein endothelial cells, by MTT assay. The IC50values at48h were7.61μM,8.29μM,8.64μM,5.30μM,4.05μM and8.11μM, respectively. It was no significant difference between the IC50of cancer cells and that of ED-25(P>0.05). It indicated that the cytotoxicity of ardisiacrispin A was non-selective. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Exposed to8μM ardisiacrispin A for48h, partial HCT-15cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase.
(3) Acute toxicity test showed ardisiacrispin A is a low-toxic compound. It's median lethal dose (LD50) was1.44g/kg. After4weeks of oral administration, the livers of the high-dose group rats were showed mild acidophilic change, cell shrinkage and loose connection. It suggested that ardisiacrispin A maight effect on liver.
(4) The in vivo antitumor activity of ardisiacrispin A per os showed that relative tumor proliferation rate AT/AC of the human colon adenocarcinoma xenograft tumors in nude mice was more than42%. It was considered as no significant anticancer activity by the Division of Cancer Treatment, NCI, NIH.
Conclusion
(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.
(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase.
(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests. Acute toxicity test showed ardisiacrispin A is a low-toxic compound.
(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was carried out, and no significant anticancer activity for oral administration was observed.
Part Two Studies on chemical constituents from the leaves of Cajanus cajan, and their anticancer effect
Background and Objective
According to our previous experiments and literatures, the stilbene-structural analogues of longistylin A, C from Cajanus cajan (L.) Millsp. exhibited significant anticancer effect. Meanwhile researches on stilbenes from Cajanus cajan were still insufficient. Therefore in order to discover new anticancer leading compounds with stibene strcture we carried out a systematic studies on isolation and structural identification of stilbenes from the leaves of Cajanus cajan, as well as their anticancer effect.
Method
(1) Stilbenes were isolated from the CHCl3extract of the leaves of Cajanus cajan by silica gel and Sephadex LH-20column chromatography. Their structures were elucidated on the basis of IR, MS and NMR spectrum, as well as elementary analysis.
(2) Effects of the isolated and identified stilbenes on cell proliferation were examined in vitro by MTT assay. The apoptosis effect on cell line was evaluated by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.
Result
(1) Seven compounds were isolated and identified as longistylin A (1), longistylin C (2), cajanstilbene H (3), cajanolactone A (4), pinostrobin (5), naringenin-4',7-dimethyl ether (6) and β-sitosterol (7). Among them cajanstilbene H (3) and cajanolactone A (4) were new compounds. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.
(2) Stilbenes1-4exhibited significant anticancer effects in vitro. New compound cajanstilbene H dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines in a MTT assay test. The IC50values at48h were21.47μM,25.83μM,21.42μM,25.85μM,24.81μM, and22.29μM, respectively. After48h of exposure to42μM cajanstilbene H, partial NCI-H460cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of NCI-H460cells, and arrest of the cell cycle at G0/G1phase.
Conclusion
(1) Two new stilbenes, cajanstilbene H and cajanolactone A, were found from the leaves of Cajanus cajan. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.
(2) Cajanstilbene H showed significant in vitro anticancer effect on NCI-H460cells. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of the cells and arrest of cell cycle at G0/G1phase.
本学位论文旨在研究发现天然抗肿瘤药物活性先导化合物。由两部分组成。
第一部分朱砂根中百两金皂苷A的分离鉴定、批量制备及抗肿瘤作用研究
研究背景和目的
作者通过前期的试验和文献调研,认为中药朱砂根(Ardisia crenata Sims.)中的三萜皂苷类化合物百两金皂苷A、B (ardisiacrispin A, B)及其结构类似物具有明显的抗肿瘤药效；鉴于文献主要集中在朱砂根皂苷的体外抗肿瘤药理研究方面,本课题对朱砂根三萜皂苷类化合物进行分离鉴定,开展毒理学和体外体内抗肿瘤活性研究,为考察百两金皂苷A类似物作为抗肿瘤药物先导化合物的可行性提供实验依据。
研究方法
(1)利用正相硅胶、MCI、反相ODS等柱层析手段,从朱砂根的正丁醇部位中分离皂苷类化合物；采用MS、1H-NMR、13C-NMR等波谱学方法鉴定化学结构。
(2)采用MTT法检测百两金皂苷A对多种人肿瘤细胞株和人非肿瘤细胞株的细胞毒性作用,利用瑞氏-吉姆萨染色法和流式细胞仪检测了百两金皂苷A对HCT-15细胞凋亡和细胞周期的影响。
(3)首次完成了化合物百两金皂苷A纯品的急性毒性和亚急性毒性研究。
(4)采用人HCT-15肿瘤裸鼠皮下移植模型,对化合物百两金皂苷A开展了体内抗肿瘤作用试验。
研究结果
(1)从朱砂根中分离得到3个化合物,其化学结构鉴定为：百两金皂苷A(ardisiacrispin A)、朱砂根皂苷B(ardisicrenoiside B)和岩白菜素(Bergenin)。分离制备了多达45g的百两金皂苷A纯品作为体内实验研究用药。
(2)体外抗肿瘤试验结果表明,百两金皂苷A对人非小细胞肺癌细胞株NCI-H460、人前列腺癌细胞株PC-3、人乳腺癌细胞株MCF-7、人宫颈癌细胞株Hela.人结直肠腺癌细胞株HCT-15、人口腔上皮癌耐药细胞株KB-V1等肿瘤细胞均具有明显的增殖抑制作用,其半数抑制浓度(IC5o)分别为7.61μM、8.29μM、8.64μM、5.30μM和4.05μM；对人肝静脉内皮细胞株ED-25的细胞毒性(IC50为8.11μM),与对上述肿瘤细胞的细胞毒性无显著差异(P>0.05),提示百两金皂苷A的细胞毒作用可能无选择性。其中,百两金皂苷A对HCT-15细胞的体外研究结果系首次发表。经细胞形态学观察发现,8μM的百两金皂苷A处理后,HCT-15细胞出现早期凋亡样形态学改变：细胞变圆,核染色质浓集,固缩,碎裂。推测其细胞毒性可能与诱导凋亡有关。Annexin V/PI双染法检测试验结果表明,随着百两金皂苷A药物浓度的增加,HCT-15细胞平均早期凋亡率逐渐上升,并呈一定的浓度依赖性。结合形态学观察结果,表明百两金皂苷A对HCT-15的细胞毒作用是通过诱导凋亡产生的。流式细胞仪检测发现,经百两金皂苷A处理可以引起HCT-15细胞发生S期阻滞。因此,百两金皂苷A可能是藉此诱导HCT-15细胞凋亡的。
(5)急性毒性试验结果显示,百两金皂苷A对昆明种小鼠的半数致死量LD50为1.44g/kg,属于低毒化合物。亚急性毒性试验中,连续灌胃4周造成了大鼠轻微肝损伤,肝细胞轻度嗜酸性变,细胞固缩,连接松散。预示着百两金皂苷A对肝脏可能有一定影响。
(6)在人HCT-15肿瘤裸鼠移植瘤模型实验中,以60mg/kg、50mg/kg、40mg/kg剂量灌胃给药百两金皂苷A,与阳性药多西他赛组相比,相对肿瘤增殖率AT/AC大于42%,根据NCI评判标准,认为该途径下百两金皂苷A对HCT-15人癌裸鼠模型的抗肿瘤药效不明显。
结论与创新点
(1)从朱砂根中分离得到3个化合物,经波谱学技术鉴定为百两金皂苷A、B,以及岩白菜素；制备了多达45g的百两金皂苷A纯品,作为体内实验研究用药。
(2)首次研究报告了百两金皂苷A对HCT-15细胞的细胞增殖抑制作用；该作用的机理与诱导肿瘤细胞凋亡和阻滞细胞增殖于S期有关。
(3)首次研究评价了百两金皂苷A的急性、亚急性毒性。结果表明,该化合物口服低毒。
(3)在人HCT-15肿瘤细胞裸鼠皮下移植模型上,测试了灌胃给药条件下百两金皂苷A的体内抗肿瘤作用,结果表明作用不明显。
第二部分树豆叶的化学成分及抗肿瘤作用研究
研究背景、目的
作者通过预实验和文献调研,认为树豆(Cajanus cajan (L.) Millsp.)叶中木豆素A、C (longistylin A, C)的二苯乙烯(菧)结构类似物有明显的抗肿瘤作用,而且目前对树豆蔗类成分的研究尚不够系统和深入。因此,课题组对树豆叶的菧类化合物进行了较为系统的分离和结构鉴定研究、体外抗肿瘤活性与机理研究,以期获得新菧类抗癌先导化合物。
研究方法
(1)利用『正相硅胶、Sephadex LH-20凝胶等柱层析手段,从树豆的三氯甲烷部位中分离蔗类化合物,并采用IR、1H-NMR、13C-NMR、2D-NMR等波谱学技术,结合EI-MS、ESI-MS和HRESI-MS等质谱和元素分析进行综合分析,鉴定化学结构。
(2)采用MTT法检测分离得到的菧类化合物对多种人肿瘤细胞株和人非肿瘤细胞株的细胞毒性作用,利用瑞氏-吉姆萨染色法和流式细胞仪检测菧类化合物对肿瘤细胞的凋亡和细胞周期的影响。
研究结果
(1)从树豆中分离得到7个化合物(1-7)。它们的化学结构经波谱学分析鉴定为：木豆素A(longistylin A)(1)、木豆素C(longistylin C)(2)、树豆菧H (cajanstilbene H)(3)、树豆内酯A (cajanolactone A)(4)等4个蔗类化合物；异美五针松双氢黄酮(pinostrobin)(5)、柚皮素4’,7-二甲醚(naringenin-4',7-dimethyl ether)(6)等2个黄酮；以及1个甾类化合物β-谷甾醇(β-sitosterol)(7)。其中3和4为新化合物,分别命名为树豆菧H (cajanstilbene H)和树豆内酯A (cajanolactone A),前者是从该属植物中首次发现的含卤素的羟基异戊烯取代二苯乙烯衍生物,后者为该属植物中首次发现的内酯化的羟基异戊烯取代二苯乙烯羧酸衍生物。
(2)四种菧类化合物树豆菧H (cajanstilbene H)、树豆内酯A (cajanolactone A)、 longistylin A、longistylin C对NCI-H460、PC-3、MCF-7、Hela、HCT-15、KB-V1等人源肿瘤细胞株的增殖表现出明显抑制作用。
其中新化合物cajanstilbene H对上述肿瘤细胞的半数抑制浓度IC5o分别为21.47μM、25.83μM、21.42μM、25.85μM、24.81μM、22.29μM;对人肝静脉内皮细胞株ED-25的IC5o为38.51μM,小于对肿瘤细胞的细胞毒性,差异有显著性(P<0.05)。
细胞形态学观察发现,经42μM的cajanstilbene H处理后,NCI-H460细胞出现早期凋亡样形态学改变：细胞变圆,核染色质浓集,核固缩,碎裂。推测其细胞毒性也可能与诱导凋亡有关。Annexin V/PI双染法检测结果表明,随着cajanstilbene H药物浓度的增加,NCI-H460细胞平均早期凋亡率逐渐上升,并呈一定的浓度依赖性。说明cajanstilbene H对NCI-H460细胞的细胞毒作用是通过诱导凋亡产生的。
经流式细胞仪检测,cajanstilbene H将NCI-H460细胞能阻滞于G0/G1期而抑制增殖。
结论与创新点
(1)从树豆叶中发现了2个新的菧类化合物,其结构系通过详细的波谱学综合分析所确定,分别命名为树豆菧H (cajanstilbene H)和树豆内酯A (cajanolactone A),前者是从该属植物中首次发现的含卤素的羟基异戊烯取代二苯乙烯衍生物,后者为该属植物中首次发现的内酯化的羟基异戊烯取代二苯乙烯羧酸衍生物。
(2)研究了新化合物树豆菧H (cajanstilbene H)、树豆内酯A (cajanolactone A)、和结构类似物longistylin A、longistylin C对NCI-H460、PC-3、MCF-7、Hela、HCT-15、 KB-V1等肿瘤细胞株的体外作用,发现新化合物具有明显的体外抗肿瘤作用。初步揭示了新化合物树豆菧H的体外抗肿瘤作用机理,发现树豆菧H可诱导NCI-H460细胞的凋亡,并使细胞周期阻滞于G0/G1期。
Malignant tumor or cancer is one of the major diseases that damage severely human health, and is the second leading cause of death in the world. Chemical medication is the major treatment method for cancer at present. Chemotherapeutic agents mainly contain synthetic and natural medicines. Many of synthetic drugs are of natural origin or derived from the structures of natural products. China is rich in medicinal plant resources, so it is a decided advantage on development of safe and effective anticancer drugs or lead compounds from medicinal herbs. The aim of this dissertation is to search natural anticancer leading compounds. It consists of two parts.
Part One Ardisiacrispin A from Ardisia crenata:Isolation, Identification, Lot Manufacture, and Anticancer Effect
Background and Objective
According to our previous experiments and literatures, ardisiacrispin A, B from Ardisia crenata Sims., as well as their derivatives, exhibited significant anticancer activity They are worthy of our further and systemaic researches, including phytochemical study, in vitro and in vivo pharmacological studies for searching of anticancer leading compounds
Method
(1) Ardisiacrispin A and relavent constituents were isolated from the n-BuOH extract of Ardisia crenata by silica gel, MCI and ODS column chromatographies. The structures were elucidated on the basis of spectroscopic analysis, mainly on MS and NMR.
(2) Effects of ardisiacrispin A on cancer cell lines were examined by MTT assay. The apoptosis was observed by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.
(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests.
(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was also carried out.
Result
(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.
(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines and cell line ED-25, human liver vein endothelial cells, by MTT assay. The IC50values at48h were7.61μM,8.29μM,8.64μM,5.30μM,4.05μM and8.11μM, respectively. It was no significant difference between the IC50of cancer cells and that of ED-25(P>0.05). It indicated that the cytotoxicity of ardisiacrispin A was non-selective. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Exposed to8μM ardisiacrispin A for48h, partial HCT-15cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase.
(3) Acute toxicity test showed ardisiacrispin A is a low-toxic compound. It's median lethal dose (LD50) was1.44g/kg. After4weeks of oral administration, the livers of the high-dose group rats were showed mild acidophilic change, cell shrinkage and loose connection. It suggested that ardisiacrispin A maight effect on liver.
(4) The in vivo antitumor activity of ardisiacrispin A per os showed that relative tumor proliferation rate AT/AC of the human colon adenocarcinoma xenograft tumors in nude mice was more than42%. It was considered as no significant anticancer activity by the Division of Cancer Treatment, NCI, NIH.
Conclusion
(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.
(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase.
(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests. Acute toxicity test showed ardisiacrispin A is a low-toxic compound.
(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was carried out, and no significant anticancer activity for oral administration was observed.
Part Two Studies on chemical constituents from the leaves of Cajanus cajan, and their anticancer effect
Background and Objective
According to our previous experiments and literatures, the stilbene-structural analogues of longistylin A, C from Cajanus cajan (L.) Millsp. exhibited significant anticancer effect. Meanwhile researches on stilbenes from Cajanus cajan were still insufficient. Therefore in order to discover new anticancer leading compounds with stibene strcture we carried out a systematic studies on isolation and structural identification of stilbenes from the leaves of Cajanus cajan, as well as their anticancer effect.
Method
(1) Stilbenes were isolated from the CHCl3extract of the leaves of Cajanus cajan by silica gel and Sephadex LH-20column chromatography. Their structures were elucidated on the basis of IR, MS and NMR spectrum, as well as elementary analysis.
(2) Effects of the isolated and identified stilbenes on cell proliferation were examined in vitro by MTT assay. The apoptosis effect on cell line was evaluated by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.
Result
(1) Seven compounds were isolated and identified as longistylin A (1), longistylin C (2), cajanstilbene H (3), cajanolactone A (4), pinostrobin (5), naringenin-4',7-dimethyl ether (6) and β-sitosterol (7). Among them cajanstilbene H (3) and cajanolactone A (4) were new compounds. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.
(2) Stilbenes1-4exhibited significant anticancer effects in vitro. New compound cajanstilbene H dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines in a MTT assay test. The IC50values at48h were21.47μM,25.83μM,21.42μM,25.85μM,24.81μM, and22.29μM, respectively. After48h of exposure to42μM cajanstilbene H, partial NCI-H460cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of NCI-H460cells, and arrest of the cell cycle at G0/G1phase.
Conclusion
(1) Two new stilbenes, cajanstilbene H and cajanolactone A, were found from the leaves of Cajanus cajan. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.
(2) Cajanstilbene H showed significant in vitro anticancer effect on NCI-H460cells. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of the cells and arrest of cell cycle at G0/G1phase.
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